The present invention relates to monoclona antibodies directed toward platinum (II) complexes, their preparation and uses such as carrying platinum (II) complexes to tumor.
Murine monoclonal antibodies directed toward antigenic determinants expressed on the surface of human tumor cells have the potential to selectively localize in tumors after systemic administration (Goldenberg et al., Science 208:1284-1286, (1980); Goldenberg et al., Cancer 45:2500-2505, (1980); Larson et al., J. Nucl. Med., 24:123-129, (1983); Mach et al., N. Engl. J. Med., 303:5-10, (1980)). This property has been successfully exploited due to recent advances in hybridoma technology which have made available large amounts of purified, high-specificity murine monoclonal antibodies directed toward a wide variety of human tumor-associated cell-surface antigens (Kohler and Milstein, Nature, 256:495-497, (1975); White et al., Cancer Res., 45:1337-1343, (1985); Morgan et al., Hybridoma, 1:27-36 (1981)).
Previous studies by Raso et al., utilizing in vitro and in vivo animal models, have demonstrated that a variety of drugs and toxins, including the A chain from the plant toxin ricin and the antitumor antibiotic agent neocarcinostatin, can be successfully targeted to human tumor cells (Raso, Immunological Rev., 62:93-117 (1982); Raso et al., Cancer Res., 41:2073-2078 (1981); Raso et al., In Receptor-Mediated Targeting of Drugs: NATO Advanced Studies Institute, Gregoriadis (ed), V 2, Plenum Press, NY (1983); Raso et al., Fed. Proc., 37:1350 (1978).
The cytotoxic potential of platinum coordination complexes was first discovered in the mid-sixties (Rosenberg et al., Nature, 205:698-699 (1965)). cis-Diaminedichloro-platinum (II) (cisplatin) was identified in experimental tumor systems as the most active of these compounds (Rosenberg, Naturwissenschaften, 60:399-406 (1973)). This unique inorganic chemotherapeutic drug is utilized as a first-line agent in the treatment of metastatic testicular and ovarian carcinomas, in combination chemotherapy for the treatment of carcinomas of the head and neck and bladder (Gale, In: Antineoplastic and Immunosuppressive Agents, Pt. II, Sartorelli et al., (eds), Handbuch der Experimentellen Pharmakologie, 38:829-840 (1975); Rosenzweig et al., Ann. Intern. Med., 86:803-812 (1977)), and is constantly introduced into new treatment protocols. The cell nucleus is thought to be the most important site for the drug's cytotoxic activity; when cisplatin enters a cell, two highly reactive ligand sites are formed as a result of chloride ion hydrolysis. These sites react with DNA to form inter- and intrastrand crosslinks which disrupt and unwind portions of the double helix (Munchausen et al., Cancer Chemother. Rep., 59:643-646 (1975 )).
The clinical use of platinum complexes is associated with a high incidence of serious, dose-limiting toxic side effects arising as a consequence of the high chemical liability and non-tumor tissue deposition of platinum complexes. Renal distal tubular damage and neurotoxic effects, including peripheral neuropathies, have been described (Dentino et al., Cancer, 4:1274-281 (1978); Bourne, Austr. J. Audiology, 6:33-80 (1984)). The severity of these side effects has provided the impetus for the synthesis of second and third generation analogues with decreased toxicity and increased aqueous solubility and stability.
One third generation compound, methylimino- diacetato-1,2-diaminocyclohexane platinum (II) (MIDP; schematically described in FIG. 2) has been shown, with multi-dose administration, to be curative in the treatment of L1210 leukemia and B.sub.16 melanoma in mice. At doses with equipotent antitumor efficacy as compared to cisplatin,, MIDP apparently does not induce renal toxicity in animal models. Additionally, cells resistant to cisplatin have been shown by others not to display cross-resistance to MIDP. Thus, MIDP appears to have an advantage over the parent platinum (II) compound, i.e., retaining antitumor efficacy and potency with the diminution of toxic side effects.
The antitumor activity of platinum complexes arises as a consequence of their chemical nature, as does a high degree of non-specific reactivity. More than 90% of cisplatin in plasma is tightly bound to plasma protein, leading to a prolonged plasma half-life of bound, inactive drug (Hill et al., Cancer Chemother. Rep., 59:647-659 (1975). The remaining unbound, highly reactive drug can participate in a number of hydrolytic and degradative reactions in plasma; the metabolites formed have greater nephrotoxic potential and decreased antitumor activity (Daley-Yates et al., Biochem. Pharmacol., 33:3063-3070 (1984)). Thus, the inherent reactivity of the platinum molecule may compromise distribution to the tumor site and, therefore, antitumor efficacy. The next logical goal in the development of effective platinum analogues and object of the present invention is to solve the problem of non-tumor associated reactivity and to increase deposition within the tumor.
A variety of murine monoclonal antibodies have been developed which recognize antigens present on the surface of human tumor cells. A number of these demonstrate minimal reactivity to normal (nonmalignant) tissues and have been utilized for tumor imaging as well as for delivery of drugs and toxins to tumor tissue (Ghose et al., Eur. J. Cancer, 11:321-326 (1975); Goldenberg et al., Science, 208:1284-1286 (1980)). Various imaging studies have indicated that tumor may accumulate two to ten times more radiolabeled monoclonal antibody than non-target tissue (Moshakis et al., Br. J. Cancer, 43:575-581 (1981); Sullivan et al., Invest. Radiol., 17:350-355 (1982)). Thus, an antitumor agent bound or coupled to a monoclonal antibody may not only exhibit increased tumor distribution but also altered clearance and systemic effects. A purpose of the delivery system of the present invention is to utilize murine monoclonal antibodies for favorably modifying the pharmacology and, tissue disposition and efficacy of platinum complexes.
There are generally two types of monoclonal antibody complexes that could be prepared for utilization in drug delivery systems. In the first, for example, a tumor targeting antibody may be directly coupled to a drug, forming an irreversible covalent complex. In the second, a monoclonal antibody capable of reversibly binding a drug may be covalently coupled to an antibody recognizing a tumor-associated antigen. The direct coupling approach has been utilized for a number of therapeutic agents such as adriamycin, vindesine and methotrexate as well as plant and bacterial toxins, including ricin A chain, pseudomonas exotoxin, pokeweed antiviral protein and gelonin (Ramakrishnan et al., Cancer Res., 44:1398-1404 (1984; Hurwitz et al/., Ann. NY Acad. Sci., 417:125-136 (1983); Embleton [.sup.3 H] Brit. J. Cancer, 47:43-50 (1983); Thorpe et al., Eur. J. Biochem., 116:447-454 (1981); Bjorn et al., Cancer Res., 45:1-8 (1985); Gallego et al., Int. J. Cancer, 33:737-744 (1984); Fitzgerald et al., J. Clin. Invest., 74:966-971 (1984)). These studies concluded that direct conjugates were efficacious both in vitro and in vivo, increasing cytotoxic agent delivery and having enhanced cytotoxicity as compared to free drug or toxin. The use of direct coupling procedures, however, may result in an adverse modification of the drug or toxin such that a significant portion of the coupled molecules are inactivated. Also, covalent modification of monoclonal antibodies may disturb antibody conformation and can lead to a decreased affinity for the binding site. The high chemical liability of platinum complexes and the finding that slight chemical modification of this agent can lead to its inactivation eliminate the desirability of using a direct covalent coupling approach for platinum (II) complex antibody delivery systems (Fitzgerald et al., J. Clin. Invest., 74:966-971 (1984); Kerrison et al., J. Chem. Soc./Chem. Comm., 27:861 (1977); Drobnik, Cancer Chemother. Pharmacol., 10:145- 149 (1983).
Many chemotherapeutic agents suffer shortcomings which limit or complicate their use. Pharmacology studies, for example, show that some agents may be: cleared rapidly from the circulation; extensively distributed to non-tumor tissue compartments; complexed irreversibly with endogenous plasma components; or metabolically inactivated by plasma or tissue enzymes (Gilman et al., The Pharmacological Basis of Therapeutics, Ed. 6, pp 1249-1313 (1980) Macmillan publishing Co., NY; Carter et al., (eds) Principles of Cancer Treatment, pp 1-951 (1982) McGraw-Hill, NY; Camiener, Biochem. Pharmacol., 16:1398-1400 (1972)). All of these dynamic events can severely reduce the amount of biologically active drug available to the tumor. The exploitation of specific monoclonal antibody binding characteristics to confer a high degree of selectivity for indiscriminately cytotoxic drugs and toxins is an attractive possibility and part of the present invention.
Antitumor agents coupled directly to monoclonal antibodies have been proposed before (Ghose et al., Cancer, 29:1398-1400 (1972); Calendi et al., Boll. Clin. Farm., 108:25-28 (1969)). They were envisioned as unique and specific protein carriers to favorably modify the pharmacology of antitumor agents such as toxic drugs by directing these agents to tumors while reducing their distribution to sites of potential toxicity (Blythman et al., Nature, 290:145-146 (1981). previous approaches to conjugating monoclonal antibodies with either toxins or chemotherapeutic agents utilized direct covalent coupling reactions or bifunctional cross-linking reagents to attach agents or toxins tightly to an antibody carrier molecule (Hurwitz et al., Ann. NY Acad. Sci., p 125 (1983); Moolten et al., Science, 169:68-70 (1970)). Problems associated with this covalent coupling approach, as mentioned above, may result from the covalent modification of murine antibodies disturbing antibody integrity and modifying antigen recognition sites of the antibody. In addition, covalent modification of some active therapeutic agents may significantly reduce or completely ablate their antitumor efficacy (Hurwitz et al., Cancer Res., 35:1175-1181 (1975); Kato et al., Cancer Res., 44:25-30 (1984). In many cases, the full expression of biological activity of cytotoxic agents may be hindered by covalent coupling to a carrier such as an antibody. For example the antiproliferative activity of such covalently altered complexes may depend on: 1) extracellular hydrolytic or enzymatic release of the active agent from the conjugate at the tumor site (Ghose et al., J. Natl. Canc. Inst., 61:657-676 (1978); or 2) internalization and intracellular cleavage of the stable bond between the active agent and the antibody (Bjorn et al., Cancer Res., 45:1-8 (1985). Hydrolytic or enzymatic release of cytotoxic agents from covalent complexes at the tumor site may not be uniformly dependable. Degradation of the complex by enzymes present in plasma may provide premature release of the active agent and thereby compromise further the therapeutic efficacy of these covalent complexes.
The utilization of highly specific antigen recognition sites of antibodies to tightly yet reversibly bind therapeutic agents in a non-destructive fashion is a potentially attractive alternative to these covalent coupling methods and is a feature of the present invention. An object of this invention is to describe a novel, tumor-targeted antibody drug delivery system consisting of a monoclonal antibody capable of reversibly binding a therapeutic agent in combination with an antibody recognizing a tumor-associated antigen. This novel approach for reversibly coupling therapeutically active agents to tumor-targeting antibodies represents a route to circumvent some of the difficulties posed by conventional covalent coupling of active agents directly to monoclonal antibodies. In this approach the monoclonal antibody delivery system contains a reversible, drug-binding site, as schematically shown in FIG. 1. This may be accomplished by producing a carrier monoclonal antibody to the active agent and coupling this carrier to a tumor-targeting antibody. Such coupling may be accomplished, for example, by using a heterobifunctional cross-linking reagent to generate an antibody-antibody linkage (Bjorn et al., Cancer Res., 45:1-8 (1985); Carlsson et al., Biochem. J., 173:723-727 (1978).
Advantages of this type of delivery complex over one involving the covalent linkage of drugs to antibodies include, for example:
1) The affinity of the complex for the active agent can be varied by selecting anti-drug antibodies with desired affinity constants (Ka), thereby subtly altering the exchange properties of the active agent with the targeting site. This should change the efficiency with which a complex delivers cytotoxic agents to target sites. Rapidly-reversible (lower affinity) complexes may be important for quick release of low molecular weight cytotoxic agents at the tumor surface to allow more rapid transport of the agent across the cell membrane and interaction at its site of action without the hindrance of an attached, high molecular weight carrier protein. Higher affinity complexes which result in slow or minimal release of active agents may be important for the delivery of toxins such as gelonin. This particular agent should remain coupled since the antibody complex acts as both a cell-targeting carrier and an internalization mechanism to allow the toxin to localize on tumor cells and to cross mammalian cell membranes for interaction with ribosomal structure, respectively (Stirpe et al., J. Biol. Chem., 255:6947-6953, (1980).
2) The active agent itself is not subjected to chemical modification. This is of exceptional importance in attempting to target cytotoxic agents such as platinum complexes, with a definitive structure activity relationship (SAR) or chemical liability.
3) Once an appropriate drug-carrier antibody is generated and the covalent carrier antibody:antibody tumor-specific cross-linking conditions have been well-defined, a large library of reversibly agent-binding tumor targeting antibody complexes may be produced. New complexes may be generated, for example, by using tumor targeting antibodies directed to different epitopes of the same cell surface antigen, antibodies to different surface antigens present on the same cell type or using antibodies to target different tumor types of interest.
The reversible drug-binding delivery system approach may allow the specific delivery of reversibly-coupled, unmodified agents such as MIDP to tumors. This type of delivery system is schematically shown in FIG. 1. Previous studies by Raso et al. have demonstrated that this non-covalent targeting approach should be effective (Raso et al., Fed. Proc., 37:1350 (1978); Raso et al., Cancer Res., 41:2073-2078 (1981); Raso, Immunological Rev., 62:93-117 (1982); Raso et al., In Receptor-mediated Targeting of Drugs: NATO Advanced Studies Institute, Gregoriadis (ed), V 2, Plenum Press, NY (1983) in press) . Neocarzinostatin, an antitumor antibiotic, and the A-chain of ricin were delivered to tumor cells in vitro by means of reversibly binding antibody:antibody complexes. The ricin A chain-binding antibody:antibody complex was found to specifically deliver ricin to antigen-bearing target cells. This ricin-bearing complex was also shown to substantially inhibit cell growth and lead to cell death. The ricin:antibody complex was found to be 5000 times more potent compared to ricin A chain alone. The in vivo pharmacokinetics of the ricin A chain:antibody complex and free ricin A chain were examined in rabbits.
Administration of ricin:antibody conjugate led to a prolongation of the beta-phase half-life for complexed ricin as compared to free ricin alone.
Further advantages of the reversible drug-binding delivery approach of the present invention as compared to directly coupled complexes may be described in regard to MIDP as follows:
(1) Much greater flexibility is possible. For example, the diaminocyclohexane ring of MIDP represents a chemically stable portion of the molecule to which antibodies may be directed. Monoclonal antibodies described herein with varying affinities for MIDP have been isolated and could be utilized for the preparation of antibody-MIDP complexes. This will allow evaluation of the relationship of drug delivery efficiency to alterations in the affinity constant for drug binding.
(2) Flexibility should also be possible in the selection of the drug to be carried by antibodies reactive, for example, to the DACH ring of MIDP. These antibodies should also recognize a variety of other third generation platinum analogues which also contain the DACH ring. Thus, a number of different platinum complexes may be evaluated for tumor delivery for each targeting antibody:carrier antibody conjugate prepared.
(3) Drug carried to the tumor by such a reversibly-binding antibody:antibody complex is not chemically modified; bound molecules may represent a future pool of free, active drug.
(4) After selection of an optimal drug-binding(carrier) antibody, conjugates with various targeting antibodies may be made. This flexibility will allow a panel of antibody complexes to be prepared, all directed towards a specific tumor type or a number of tumor types.
(5) The activity of direct covalently-coupled complexes may be highly dependent on the extent of drug-antibody cell surface receptor internalization. Reversible drug-binding complexes may act quite differently; free, active drug may also be released and be available for interaction with the cell surface. Evidence is steadily accumulating that the process of antibody-binding to the cell surface may alter membrane conformation; sensitization to subsequently administered therapeutic agents may ensue (Greenfield et al., Proc. Amer. Assoc. Cancer Res., 26:336 (1985); Webb et al., Proc. Amer. Assoc. Cancer Res., 26:272 (1985)). Free MIDP, released from antibody complexes at or near the cell surface, may act in this manner. Therefore, the reversible drug-binding antibody complexes of the present invention may represent a first step in the development of a new class of novel chemotherapeutic agents with multiple mechanisms and sites of action. Further studies with MIDP should provide insights into the optimization and future utilization of this approach for the delivery of other therapeutic agents, biological modifiers and toxins.
A broad object of the present invention is to utilize a novel, specific monoclonal antibody to reversibly bind therapeutic platinum (II) complex. Other potential uses of this antibody also include but are not limited to:
1) A composition in which a reversible-binding antibody is utilized to extend the short plasma half-life and therapeutic action of a Pt complex.
2) A composition in which an antibody specific for a Pt (II) complex itself or a specific Pt (II) complex is utilized in an enzyme-linked immune-absorbent (ELISA) assay for quantitation of platinum or platinum complex in urine, serum, other biological fluids or tissues.
3) The use of antibodies specifically binding platinum (II) complex antibodies as specific probes to detect platinum complexes at the cellular or sub-cellular level.
Many other objects and advantages of the present invention too numerous to list herein are inherent in the presently described invention.